The invention relates to competitive immunoassays. More specifically, the invention relates to the use of antiidiotypic monoclonal antibody reagents as a substitute for labeled antigen in competitive immunoassays. The invention also relates to the idiotypic monoclonal antibody from which the antiidiotypic monoclonal antibody reagents are derived and to the competitive immunoassays to which these reagents are applied.
Competitive immunoassays provide a sensitive method for the detection and measurement of antigenic substances. These immunoassays usually employ one or more antigens, antibodies, and immunochemical labels arranged in a variety of configurations. Typically, a competitive immunoassay will utilize a labeled ligand (Ag*), which may be either a labeled hapten or a labeled macromolecular antigen. During the assay, the labeled ligand competes with the corresponding unlabeled antigen or analyte (Ag) from sample for a limited number of antibody binding sites (Ab). A review of many aspects of immunoassays by numerous authors is compiled in Enzyme Immunoassay, E. Maggio, Ed. CRC Press, Boca Raton, Fla. (1980). ##EQU1##
The concentration of antibody binding sites available to bind the labeled ligand is inversely related to the concentration of unlabled ligand (analyte). Measurement of the antibody-bound labeled component, or, alternatively, the unbound labeled component allows the user to relate the signal obtained from the label to the concentration of analyte of interest.
Competitive immunoassays may be either heterogeneous or homogeneous. Heterogeneous immunoassays require a physical separation of the free (unbound) labeled antigen from the antibody-bound labeled antigen. The physical separation of the free and bound labeled antigen enables a measurement of the disposition of the labeled antigen in a competitive immunoassay. Typical labels used in heterogeneous immunoassays include radioactivity, enzyme activity, fluorescence, luminescence, and the like. U.S. Pat. No. 3,709,868 is exemplary of a heterogeneous radioimmunoassay.
Many of the parameters of a suitable separation technique for heterogeneous assays are shared by enzyme, fluorescence, luminescence and radioimmunoassay procedures. In order to maximize precision and sensitivity, one would like to insure complete separation of the free and bound reactions with relatively simple and foolproof manipulations. The separation should be accomplished rapidly, preferably without elaborate or expensive equipment. In addition, an ideal method should be unaffected by the constituents of the sample (serum, plasma, cerebrospinal fluid, urine, saliva, and so forth), be generally applicable to a wide variety of analytes, and be amenable to automation.
The most common methods of separation used for enzyme immunoassay include:
I. Solid-Phase methods
A. Microtitration plates PA2 B. Coated tubes or beads PA2 C. Suspendable microbeads PA2 A. Second-antibody method
II. Immunological Precipitation
The suspendable microbeads and secondary antibody precipitation each require a centrifugation step to effect a physical separation. The centrifugation step is an impediment to automation of the technique. On the other hand, no centrifugation is required when the physical separation is effected by the employment of microtitration plates and coated tubes or beads. The avoidance of a centrifugation step greatly facilitates automation of the assay procedures since the remaining steps of these methods are simple liquid handling steps, i.e., pipetting, diluting, and washing.
Homogeneous immunoassays are used where the binding of antibody to antigen can be shown to directly modulate the signal generated by the label. Homogeneous immunoassays require no separation of the antibody-bound and free labeled components. Typical labels used in homogeneous immunoassays include radionuclides (radioimmunoassays), enzymes (enzyme immunoassays), and fluorophores (fluoroimmunoassays). U.S. Pat. No. 3,817,837 is exemplary of a homogeneous enzyme immunoassay. Less frequently used labels include chemiluminigenic molecules, latex particles, stable free radicals, lytic bacteriophage, tanned red blood cells, gold sol suspensions, and enzyme cofactors or inhibitors. U.S. Pat. No. 4,220,450 is exemplary of a chemiluminescence immunoassay.
The above mentioned assays use polyclonal antibodies and a variety of signal generating labels in various types of immunoassay procedures. U.S. Pat. No. 4,376,110 (David) is exemplary of an immunoassay employing monoclonal antibodies in ways similar to which polyclonal antibodies are used in the above inventions. The David immunoassay employs two idiotypic monoclonal antibodies which compete for antigen In the present invention, an antiidiotypic monoclonal antibody competes with antigen for binding sites on the idiotypic antibody. The present use of monoclonal antiidiotypic antibodies in conjunction with idiotypic antibodies or receptors in immunoassays is an entirely new development in the immunoassay field.
Competitive immunoassays require labeled antigen as a essential component of the reagents, i.e the labeled antigen competes with the unlabeled antigen present in sample for a limited number of antibody binding sites. While many antigens may be inexpensive and readily available, other antigens of interest are difficult to obtain or are expensive. Human antigen purified from vital body tissues (e.g., human heart, brain, muscle, nerve tissue, and the like) can be difficult to obtain. Other antigens may have a naturally low prevalence of occurrence or may be intrinsically labile and subject to denaturation, or chemically unstable. For example, certain esterase inhibitors used as neurotoxins or insecticides are subject to hydrolysis upon storage in aqueous media.
Prior to the present invention, competitive immunoassay techniques were disfavored for the analysis of difficult to obtain antigens. However, the present invention teaches that the labeled antigen can be replaced with labeled antiidiotypic monoclonal antibodies having a structural congruence with the rare or otherwise intractable antigen. The present invention enables the use of competitive immunoassys as a practical means to detect rare or otherwise intractable antigens.
An important class of antigens of particular relevance to the present invention consists of complex macromolecular and poly-macromolecular material; subcellular components such as microsomes, organelles, chromosomes and the like; viral capsids, viral nucleic acids or nucleoprotein complexes or intact viruses; cell membranes; hormone receptors and other structures which occur naturally in biological systems or which, like microsomes, are artifactually generated from structures which occur naturally in biological systems. In the past it has been difficult to produce labeled reagents in a reproducible manner in order to assay for the presence of such materials because of 1.) the variable composition of preparations of such materials, 2.) the colloidal nature associated with preparing soluble extracts of such materials; and many other similar problems.
The present invention provides for the replacement of these difficult to obtain or impossible to use antigens with antiidiotypic antibodies exhibiting congruence of structure with one or more epitopes of the difficult to use antigens; which antiidiotypic antibodies are soluble, easy to use replacement materials. The use of such antiidiotypic antibodies in assays for complex antigen materials thus affords a heretofore unavailable means of performing a group of immunoassays important to the field of clinical and analytical chemistry.
In developing an immunoassay, there are many considerations. One consideration is the signal response to changes in the concentration of analyte. A second consideration is the ease with which the protocol for the assay may be carried out. A third consideration is the variation in interference from sample to sample. Ease of preparation and purification of reagents, availability of equipment, ease of automation, and interaction with ligands, are additional considerations. The assay configuration determines the allowable user protocol(s), assay performance parameters such as sensitivity, specificity, inter- and intra-assay precision, and the speed with which results may be obtained. In assessing the practical usefulness of any of the new immunoassay methodologies, it is particularly informative to examine the assay configuration employed in the assay.
There is a continuing need for new and accurate techniques which can be adapted for a wide spectrum of different ligands which can be used in specific cases where other methods may not be readily adaptable. The present invention advances the immunoassay field by its use of antiidiotypic monoclonal antibodies in conjunction with idiotypic antibodies or receptors.